1. Field of the Invention
The present invention is directed to substantially pure protein-polymer conjugates. In particular, the invention is directed to histidine-linked protein-polymer conjugates and methods of making the same.
2. Description of Related Art
Conjugating biologically-active proteins to polymers has been suggested to improve one or more of the properties of circulating life, water solubility or antigenicity in vivo For example, some of the initial concepts of coupling peptides or polypeptides to polyethylene glycol (PEG) and similar water-soluble polymers are disclosed in U.S. Pat. No. 4,179,337, the disclosure of which is incorporated herein by reference.
Insulin and hemoglobin were among the first therapeutic agents conjugated. These relatively large polypeptides contain several free lysine .epsilon.-amino attachment sites. Several polymers could be attached without significant loss of biologic activity.
For many biologically active materials, the conjugation process, however, is not without complications. The conjugation process is not specific with regard to attachment sites. Care must be taken to limit the loss of biological activity caused by the conjugation reaction. For example, if too much of the activated polymer is attached to the target protein or polypeptide, biological activity can be severely reduced or lost. Further, if the wrong linker joining the polymer to the protein is used or if an insufficient amount of polymer is attached to the target, the therapeutic value of the resultant conjugate is rather limited. Often, such conjugates do not demonstrate enough of an increase in the circulating life to compensate for the loss in bioactivity. Problems can also result when a therapeutic moiety's active site (i.e. where groups associated with bioactivity are found) becomes blocked as a result of the polymer attachment. This problem can be difficult to avoid since the polymer and protein are typically joined in solution-based reactions and the polymer conjugation process is not specific with regard to attachment sites. Pre-blocking the active sites with materials such as pyridoxal phosphate has been suggested, but the results have been inconsistent. Lysine-depleted variants of proteins have also been suggested as a way of controlling polymer attachment. This technique, however, is often impractical since it adds significantly to the cost of the final product. The problems are particularly acute with lower molecular weight proteins and peptides. These bioactive materials often have few attachment sites not associated with bioactivity.
In another attempt to avoid the loss of bioactivity following polymer conjugation, granulocyte colony stimulating factor ("G-CSF") was conjugated to mPEG carboxymethyl-N-hydroxy-succinimidyl ester then treated with two molar hydroxylamine (pH 7.3) to remove "unstable" linkers, followed by a pH reduction to 3.5. Kinstler et al., 1996, Pharmaceutical Res. 13(7): 996-1002. No description or suggestion of attaining improved G-CSF nor guidance regarding treatment of any other protein conjugates was provided.
Interferons, hereinafter also referred to as IFN's, are a particular example of proteins which could benefit from improved polymer conjugation techniques. See, for example, U.S. Pat. Nos. 4,766,106 and 4,917,888 which describe inter alia beta interferon conjugated with activated polymers including mPEG-2,4,6-trichloro-S-triazine, mPEG-N-succinimidyl glutarate or mPEG-N-succinimidyl succinate. The patentees disclose that covalent modification of the protein is done at a pH of from 5 to 9 and, when the protein is reacted through its lysine residues, covalent modification of the protein is done at a pH of from 8 to 9. Relatively high molar excesses (10, 20 and 50-fold) of the activated polymer are also used.
European Patent Application bearing publication No. 0 236 987 describes reacting alpha and gamma interferons with high molar excesses of alkyl imido ester-activated polyethylene glycols under conditions which preferably include a pH of from approximately 7 to 9. European Patent Application bearing publication No. 0 510 356 describes conjugating alpha interferon with pyridinyl carbonyl and thiocarbonyl activated PEG at a pH of from 7 to 9. There was no mention in these disclosures that amino acids other than lysine were involved in the conjugation or that it would be advantageous to do so.
WO96/11953 reports that conjugates were prepared by reacting a protein, exemplified by consensus IFN, with a polymer, at an acid pH (pH 4) using a reductive alkylation reaction for the selective attachment of polymer, e.g., PEG, to the N-terminal. WO96/11953 states that this reaction selectively prevents linkage to lysine epsilon amino groups, while favoring linkage with the N-terminal alpha amino group. WO96/1 1953 also describes a two-step pH treatment process wherein G-CSF is reacted with a PEG at pH 8.0, followed by reduction of pH to pH 4.0, simply as a prelude to loading the product onto a separation column. WO96/11953 does not teach or suggest the advantages of an acylation reaction to selectively attach polymers to IFN residues other than the N-terminal or lysines.
In view of the above-described disclosures, it is believed that additional improvements in interferon-polymer conjugates are desirable in order to address various shortcomings. The present invention provides additional improvements to the field and thus addresses these shortcomings.